Over the last two decades, there has been a rapid development in nucleic acid analysis, specifically in the field of nucleic acid amplification and DNA sequencing technology, with an increasing range of instrumentation now available. The conventional methods of detecting and analysing a nucleic acid sequence primarily rely on fluorescent nucleic acid intercalating dyes, fluorescent-labelled oligonucleotide probes, fluorescent- or radioactive-labelled nucleotides.
Subsequently, a new method of analysing nucleic acid synthesis and sequencing has been developed using a semiconductor-based detection system such as an Ion Sensitive Field Effect Transistor (ISFET), see for instance our PCT publication WO 03/073088. An Ion Sensitive Field Effect Transistor-based platform, unlike conventional fluorescent-based nucleic acid analysis systems, does not require expensive optical instruments or dangerous radioactive isotopes for detection, thus making this platform a cost effective, safe and simple alternative for sequencing and nucleic acid amplification analysis.
Specifically, an ISFET, which measures ion concentrations in solution, has been employed to detect nucleotide incorporation into a nucleic acid strand by detecting the change in hydrogen ion (H+, proton) concentration resulting from the reaction.
Hydrogen ions are released during the nucleic acid polymerization reaction. For example, Equation I below demonstrates the release of hydrogen ion facilitated by DNA polymerase mediated hydrolysis of a single deoxynucleotide:dNTP→dNMP+PPi+zH+  (Equation I)wherein dNTP is a nucleoside triphosphate, dNMP is a nucleoside monophosphate, z is an integer or fraction describing the average number of protons generated per nucleotide turnover, H+ is a proton and PPi is a Pyrophosphate (leaving group or reaction product).
The reaction can be driven to further produce more hydrogen ions by hydrolysing the pyrophosphate into two orthophosphates (Pi). Such a secondary chemical reaction is facilitated by pyrophosphatases and is depicted in Equation II:PPi→2Pi+zH+  (Equation II)
The workflow of current ‘sequencing-by-synthesis’ methodology can be broadly divided into template preparation, sequencing and detection, and data analysis. The first step, template preparation, usually involves clonally amplifying the template in order to achieve sufficient quantity of amplified template to confidently detect nucleotide incorporation signal during sequencing step. Currently, such clonal amplification step is usually performed in a compartment separate from the sequencing reaction and typically in a separate machine. However, such spatial separation of the two steps requires a skilled work force, high levels of hands on time, introduces incremental errors and may increase sample loss, thus decreasing the sensitivity of detection for sequencing and increasing costs.
To facilitate accurate sequencing, the standard practice is to amplify the nucleic acid. Various methods for amplifying nucleic acid are known. Indeed, PCT publication (WO2008/107014) discloses a method of monitoring qPCR using Solid-State pH Sensing, for instance an ISFET. Reaction monitoring is by means of detecting a change of pH resulting from proton release in the presence of a target (nucleic acid) sequence as amplification proceeds beyond a threshold number of cycles for the buffering capacity of a sample to be overcome. It does not disclose sequence detection via sequencing-by-synthesis, only detection of the amplification activity itself. WO2008/076406A2 also discloses various methods of amplification, such as bridge amplification, in the context of ISFETs.
Sequencing methods using ISFETs for determining are also known. US 2010/031398A1 discloses an apparatus for use in a method of sequencing, the apparatus comprising an array of microwells and sensors, which may be ISFETs. The sensors have a floating gate structure which in turn has a layer of protection material disposed over the floating gate from the analyte. The protection material has a thickness of up to about 600 Angstroms. Methods of manufacture are also provided. However, these sequencing methods operate as a distinct process on pre-prepared [colonies] of DNA.
Thus, it is an object of the present invention to provide an ion sensitive apparatus for amplification and sequencing, and a method for amplifying and sequencing nucleic acids which overcomes or mitigates the disadvantages posed by the existing methods of sequencing.
It is a further object of the invention to provide an ion sensitive apparatus for amplification and sequencing which overcomes the difficulties arising from the need to combine both amplification and subsequent sequencing without any spatial separation.